HPIV serotypes 1, 2, and 3 are significant causes of severe respiratory tract disease in infants and young children worldwide. The HPIVs are enveloped, non-segmented, negative strand RNA viruses of the family Paramyxoviridae. The broad outlines of their biology and molecular genetics have been defined in previous studies by this laboratory and others. The HPIV genome encodes three nucleocapsid-associated proteins, namely the nucleoprotein N, phosphoprotein P, and large polymerase protein L; and three envelope-associated proteins, namely the internal matrix protein M and the fusion F and hemagglutinin-neuraminidase HN transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and the major protective antigens. In addition, the P gene encodes various accessory protein(s)from one or more additional ORFs: C (HPIV1), V (HPIV2), and C, D, and possibly V (HPIV3). These accessory proteins have a number of functions that antagonize the host response to viral infection, as described in previous years. We are developing attenuated versions of HPIV1, 2, and 3 that also express the fusion F protein of human respiratory syncytial virus (RSV). As noted, RSV is the most important viral agent of severe pediatric respiratory tract disease, with a contribution to human disease comparable to that of the HPIVs combined, and the F protein is the major RSV neutralization and protective antigen. HPIV1, 2, and 3 expressing the RSV F protein would provide bivalent vaccines against each respective HPIV and RSV. There are several advantages to an HPIV-vectored RSV vaccine compared to an attenuated RSV strain. The HPIVs replicate more efficiently in cell culture than RSV and, whereas RSV is prone to physical instability and loss of infectivity, the HPIVs are relatively stable. They also form spherical particles compared to the large filaments of RSV, making them more amenable to filtration and other steps in manufacture. Taken together, these attributes make HPIV vectors much easier to manufacture, distribute, store, and use compared to attenuated RSV strains, and this advantage may be essential for extending RSV vaccines to resource-challenged countries. Furthermore, our studies in experimental animals suggest that boosting RSV responses is more efficient using HPIV/RSV vectors as opposed to boosting with attenuated RSV strains, since the latter are subject to greater restriction by prior RSV-specific immunity. We are working on vectors from each of the three serotypes, but are emphasizing HPIV3 for several reasons. As noted, HPIV3 is more suitable for immunization early in infancy because it infects and causes disease at that time, and thus it would be appropriate to vaccinate simultaneously against RSV and HPIV3. In contrast, HPIV1 and HPIV2 tend to infect after one year of life, and those vectors may be more suitable for use at that later time. In addition, HPIV3 is the second most important viral agent of serious pediatric respiratory tract disease, following RSV, and thus a combined HPIV3/RSV vaccine would address the two most important viral agents of severe respiratory tract disease. Also, we previously developed an attenuated HPIV3 platform that appears to be an excellent vector candidate. This virus is called B/HPIV3 and consists of bovine PIV3 in which the F and HN genes have been replaced by those of HPIV3, yielding a chimeric virus that is attenuated in primates due to the bovine backbone but which bears the neutralization and major protective F and HN antigens of HPIV3. Furthermore, B/HPIV3 has been evaluated in clinical phase 1 studies, both as an empty vector (LID/NIAID study) and as a vector for RSV-F (MedImmune study), and was shown to be well-tolerated in either role in infants and young children. In the initial clinical study of B/HPIV3-RSV-F, the RSV F insert exhibited substantial instability and was not as immunogenic as hoped. We believe that the stability of the vector can be improved by more careful preparation of the vaccine material, and that the immunogenicity of the RSV F insert can be improved by better vector design. These are immediate objectives. We are evaluating a number of parameters with regard to vector design and the expression of the RSV F foreign gene from attenuated versions of HPIV1, 2, and 3. Most of this work is still in progress and will be discussed in the 2015 report. As noted, B/HPIV3 is the first priority. At the present time, we have completed evaluation of the effects of the position of the RSV F insert in the B/HPIV3 backbone. Specifically, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the 1st (pre-N), 2nd (N-P), 3rd (P-M), and 6th (HN-L) genome positions. Insert position potentially can have several effects. First, expression of the RSV F insert would be expected to be greatest when the insert is placed closer to the viral promoter at the 3' end of the genome, because promoter-proximal genes are expressed more efficiently than promoter-distal genes. However, higher expression of the RSV F protein might be deleterious to the vector because the highly fusogenic nature of RSV F might cause accelerated and enhanced cytopathogenicity. This might inhibit vector replication, and might exert a selective pressure for loss of expression of the RSV F insert. Another possible effect is that the presence of the insert might decrease the expression of downstream vector genes, and placement of the insert closer to the promoter would affect a larger number of vector genes and thus might be more attenuating. We found that there was up to a 69-fold gradient (decreasing) in RSV F expression from the 1st to the 6th position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters. This did not appear to be influenced by the level of RSV F expression and syncytium formation: this was surprising because the high levels of expression of RSV F from the 1st and 2nd positions resulted in rapid and extensive syncytium formation in cell culture yet, this did not appear to inhibit vector replication. Some of the inserts conferred temperature sensitivity. However, there was no obvious pattern to this effect. For example, insertion of the RSV F insert in the first position, which resulted in the greatest expression of RSV F and affected the greatest number of vector genes, did not confer temperature-sensitivity whereas insertion in downstream positions, which gave lower levels of RSV F expression and affected fewer vector genes, conferred temperature sensitivity. The temperature sensitive phenotype was greatest for the insert in the third position, and why the effect would be greatest in this position remains unexplained. Each B/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for positions 1 and 2 than for 6, consistent with the observed greater expression. Evaluation of insert stability suggested that RSV F is under mild selective pressure to be silenced during vector replication in vitro and in vivo. We had anticipated that instability would be enhanced by increased expression of RSV F, but this was not what was observed: rather, stability was greater for positions 1 and 2 despite high levels of RSV F expression. In any event, the amount of instability observed in vitro was minor, involving a small percentage of recovered vector. Instability was increased during replication in hamsters, but still involved a small percentage of vector. These findings suggest that stability and expression can be increased for the B/HPIV3-RSV-F vector. These findings will be augmented by studies of additional factors presently in progress. These findings should be generally applicable to vectors based on HPIV1 and HPIV2.